Chondroitin sulfate synthesis promoter

ABSTRACT

The invention provides a chondroitin sulfate synthesis promoter useful for the treatment of diseases such as articular disease and discopathy. The chondroitin sulfate synthesis promoter contains, as an active ingredient, chondroitin sulfate glucuronyltransferase protein and/or chondroitin sulfate N-acetylgalactosaminyltransferase-1 protein, or a gene encoding the enzyme protein(s).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Japanese application number2006-045813, filed on Feb. 22, 2006 and Japanese application number2006-224252, filed on Aug. 21, 2006, both of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a chondroitin sulfate synthesispromoter, and to a treatment agent for articular disease and discopathyemploying the promoter.

2. Background Art

Cartilage contains a macromolecular substance, called aggrecan, which ispredominantly formed of chondroitin sulfate proteoglycan. Aggrecan is atype of proteoglycan in which a polysaccharide sulfate is covalentlybound to a core protein, and is known to play an important role inorganisms. In cartilage, aggrecan, having a hyaluronic acid-bindingdomain in a core protein portion, forms a macromolecular complex with alink protein and hyaluronic acid. Aggrecan, having 100 or morechondroitin sulfate chains bound to a core protein portion thereof,retains a large number of water molecules. Thus, aggrecan is present incartilage in the form of highly hydrated gel and is believed to playimportant roles such as absorbing shock and reducing friction(lubrication).

Meanwhile, several glycosyltransferases have recently been reported tobe involved in synthesis of chondroitin sulfate chains: chondroitinsynthase-1 (CSS-1, J. Biol. Chem., October 2001; 276: 38721-38726),chondroitin synthase-2 (CSS-2, J. Biol. Chem., August 2003; 278:30235-30247, WO 03/102194), chondroitin synthase-3 (CSS-3, J. Biol.Chem., October 2003; 278: 39711-39725, WO 03/102193), chondroitinsulfate glucuronyltransferase (CSGlcAT, J. Biol. Chem., October 2002;277: No. 41: 38179-38188), chondroitin sulfateN-acetylgalactosaminyltransferase-1 (CSGalNAcT-1, J. Biol. Chem., March2002; 277: No. 11: 8841-8846), and chondroitin sulfateN-acetylgalactosaminyltransferase-2 (CSGalNAcT-2, J. Biol. Chem.,January 2003; 278: 3063-3071, Japanese Patent Application Laid-Open(kokai) No. 2003-289883).

Although general functions of these enzymes are already known, the rolesof these enzymes in organisms have not yet been elucidated. In addition,an enzyme involved in biosynthesis of chondroitin sulfate (a part ofaggrecan biosynthesis) has not been identified. Therefore, whether ornot these enzymes increase the amount of chondroitin sulfate inorganisms remains unknown.

SUMMARY OF THE INVENTION

Degradation of cartilage is accelerated by aging or overload. Forexample, in recent aging society, an increased number of elderly peoplesuffer knee joint pain caused by wear of knee cartilage, leading to keendemand for means of repairing cartilage functions. In cartilage ofelderly people, shock absorption performance and friction reductionperformance (lubricating action) are considerably impaired. Loss offunctions intrinsic to cartilage may predominantly be attributable to adecrease in the amount of chondroitin sulfate chains contained byproteoglycan such as aggrecan. Under such circumstances, the presentinventors have attempted to repair and improve functions of cartilagethrough promoting biosynthesis of chondroitin sulfate chains ofaggrecan.

In order to solve the aforementioned object, the present inventors haveconducted extensive studies, and have found that among chondroitinsulfate synthesis-related enzymes, chondroitin sulfateglucuronyltransferase (hereinafter sometimes abbreviated as CSGlcAT) andchondroitin sulfate N-acetylgalactosaminyltransferase-1 (hereinaftersometimes referred to as CSGalNAcT-1) are expressed in mouse embryoniccartilage and mouse cartilage differentiated cells in an expressionpattern which is considerably correlated with a known aggrecanexpression pattern. The inventors have also found that forcedover-expression of CSGlcAT and CSGalNAcT-1 in chondroid cells remarkablypromotes synthesis of chondroitin sulfate. The inventors have furtherfound that introduction of CSGalNAcT-1 Gene to mouse intervertebraldiscs increases the levels of chondroitin sulfate in the intervertebraldiscs, through in vivo experiment.

The present invention has been accomplished on the basis of thesefindings.

Accordingly, in one aspect of the present invention, there is provided achondroitin sulfate synthesis promoter comprising, as an activeingredient, a gene encoding chondroitin sulfate glucuronyltransferaseand/or a gene encoding chondroitin sulfateN-acetylgalactosaminyltransferase-1.

Preferably, the gene encoding chondroitin sulfate glucuronyltransferaseis a nucleic acid molecule selected from at least one of the following:

(a): a nucleic acid molecule including a nucleotide sequence defined bySEQ ID NO: 1,

(b): a nucleic acid molecule which can be hybridized with a complementof a nucleic acid of SEQ ID NO: 1 under stringent conditions, or

(c): a nucleic acid molecule including a nucleotide sequence identicalto that defined by SEQ ID NO: 1, except that one or more nucleotide(s)have been substituted, deleted, inserted, or transposed, wherein thenucleic acid molecule encodes a protein having activity of transferringglucuronate from a glucuronate donor to a non-reducing endN-acetylgalactosamine residue present in the chondroitin skeleton.

Preferably, the chondroitin sulfate synthesis promoter comprises, as anactive ingredient, a nucleic acid molecule including a nucleotidesequence encoding an amino acid sequence defined by SEQ ID NO: 2.

Preferably, the gene encoding chondroitin sulfateN-acetylgalactosaminyltransferase-1 is a nucleic acid molecule selectedfrom at least one of the following:

(d): a nucleic acid molecule including a nucleotide sequence defined bySEQ ID NO: 3,

(e): a nucleic acid molecule which can be hybridized with a complementof a nucleic acid of SEQ ID NO: 3 under stringent conditions, or

(f): a nucleic acid molecule including a nucleotide sequence identicalto that defined by SEQ ID NO: 3, except that one or more nucleotide(s)have been substituted, deleted, inserted, or transposed, wherein thenucleic acid molecule encodes a protein having activity of transferringan N-acetylgalactosamine residue from an N-acetylgalactosamine donor toa non-reducing end D-glucuronate residue of an N-acetylgalactosamineacceptor substrate containing a sugar chain represented by formula;

GlcUA-Gal-Gal-Xyl

(wherein GlcUA represents a D-glucuronate residue, Gal represents aD-galactose residue, Xyl represents a D-xylose residue, and “-”represents a glycosidic linkage).

Preferably, the chondroitin sulfate synthesis promoter comprises, as anactive ingredient, a nucleic acid molecule including a nucleotidesequence encoding an amino acid sequence defined by SEQ ID NO: 4.

In another aspect of the present invention, there is provided achondroitin sulfate synthesis promoter comprising, as an activeingredient, chondroitin sulfate glucuronyltransferase and/or chondroitinsulfate N-acetylgalactosaminyltransferase-1.

Preferably, the chondroitin sulfate glucuronyltransferase is an enzymeselected from at least one of the following:

(A): an enzyme comprising a protein which includes an amino acidsequence defined by SEQ ID NO: 2, or

(B): an enzyme comprising a protein which includes an amino acidsequence identical to that defined by SEQ ID NO: 2, except that one ormore amino acid residue(s) have been substituted, deleted, inserted, ortransposed, wherein the protein has activity of transferring glucuronatefrom a glucuronate donor to a non-reducing end N-acetylgalactosamineresidue present in the chondroitin skeleton.

Preferably, the chondroitin sulfate N-acetylgalactosaminyltransferase-1is an enzyme selected from at least one of the following:

(C): an enzyme comprising a protein which includes an amino acidsequence defined by SEQ ID NO: 4, or

(D): an enzyme comprising a protein which includes an amino acidsequence identical to that defined by SEQ ID NO: 4, except that one ormore amino acid residue(s) have been substituted, deleted, inserted, ortransposed, wherein the protein has activity of transferring anN-acetylgalactosamine residue from an N-acetylgalactosamine donor to anon-reducing end D-glucuronate residue of an N-acetylgalactosamineacceptor substrate containing a sugar chain represented by formula;

GlcUA-Gal-Gal-Xyl

(wherein GlcUA represents a D-glucuronate residue, Gal represents aD-galactose residue, Xyl represents a D-xylose residue, and “-”represents a glycosidic linkage).

Preferably, the gene encoding chondroitin sulfate glucuronyltransferaseand a gene encoding chondroitin sulfateN-acetylgalactosaminyltransferase-1 are incorporated into an expressionvector or a host cell.

In yet another aspect of the present invention, there is provided atreatment agent for articular disease and discopathy employing, as anactive ingredient, any of the chondroitin sulfate synthesis promoters.

The chondroitin sulfate synthesis promoter according to the presentinvention can promote synthesis of chondroitin sulfate contained inproteoglycan such as aggrecan. Therefore, the synthesis promoter issuitable for improving cartilage functions and treating articulardisease and discopathy.

Accordingly, the present invention provides a method for promotingchondroitin sulfate synthesis in a living subject wherein the livingsubject includes a joint, a cartilage, an intervertebral disc or a cell,the method comprising the steps of:

providing a gene comprising a gene encoding chondroitin sulfateglucuronyltransferase or a gene encoding chondroitin sulfateN-acetylgalactosaminyltransferase-1; and

introducing the gene into the living subject.

Preferably, the step of introducing the gene into the living subject isperformed with a gene gun or via injection.

Further, the present invention provides a method for promoting achondroitin sulfate synthesis in a living subject wherein the livingsubject includes a joint, a cartilage, an intervertebral disc or a cell,the method comprising the steps of:

providing a chondroitin sulfate glucuronyltransferase or chondroitinsulfate N-acetylgalactosaminyltransferase-1; and

introducing a chondroitin sulfate glucuronyltransferase or chondroitinsulfate N-acetylgalactosaminyltransferase-1 into the living subject.

Preferably, the step of introducing a chondroitin sulfateglucuronyltransferase or chondroitin sulfateN-acetylgalactosaminyltransferase-1 into the living subject is performedvia injection.

Further, the present invention provides a method for treating anarticular disease or discopathy, the method comprising the steps of:

applying a therapeutically effective amount of an active ingredientcomprising a chondroitin sulfate glucuronyltransferase, a gene encodingchondroitin sulfate glucuronyltransferase, a chondroitin sulfateN-acetylgalactosaminyltransferase-1, and/or a gene encoding achondroitin sulfate N-acetylgalactosaminyltransferase-1 with theaffected joint or intervertebral disc; and

promoting a chondroitin sulfate synthesis in the affected joint orintervertebral disc.

These and other objects/aspects of the present invention will becomeapparent in light of the present specification, claims and drawingsappended hereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows in situ hybridization results to confirm expression ofchondroitin sulfate glycosyltransferases in cartilage (photographs);

FIG. 2 shows RT-PCR results representing expression patterns ofchondroitin sulfate glycosyltransferases during differentiation of ATDC5cells;

FIG. 3 shows RT-PCR results representing expression patterns ofchondroitin sulfate glycosyltransferases during differentiation of N1511cells;

FIG. 4 shows expression levels of chondroitin sulfateglycosyltransferases in ATDC5 cells, N1511 cells, and mouse embryonicfibroblasts (MEFs);

FIG. 5 shows expression levels of CSGlcAT and CSGalNAcT-1 in transfectedcells;

FIG. 6 shows results of Alcian Blue staining and immunostaining(employing anti-chondroitin 4-sulfate (C4S) antibody or anti-aggrecanantibody) of control (Mock) and sample of transfected cells(photographs);

FIG. 7 shows chondroitin sulfate synthesis activities of control (Mock)and sample of transfected cells as determined by labeled chondroitinsulfate levels in culture media and cell lysates, wherein “-” denotes notreatment, “HSase” denotes heparitinase mixture treatment, and “CHase”denotes chondroitinase ABC treatment (lower graphs);

FIG. 8 shows results of disaccharide analysis of GAG digested productsformed through chondroitinase ABC treatment of control (Mock) and sampleof transfected cells, the disaccharide composition being determined inculture media and cell lysates;

FIG. 9 shows results of Alcian Blue staining of intervertebral disc intransgenic mice for CSGalNAcT-1 (photographs), wherein the photographsof a, c and c are for the mock, the photographs of b, d and f are forthe transgenic mice, a and b are the overall view for the intervertebraldisc, c and d are closeups for the nucleus pulposus cells and e and fare closeups for the chondrocytes; and

FIG. 10 shows levels for alcian blue stain of intervertebral disc intransgenic mice for CSGalNAcT-1.

BEST MODE FOR PRACTICING THE INVENTION

While this invention is susceptible of embodiment in many differentforms, there is shown in the drawings and will be described in detail,several specific embodiments with the understanding that the presentdisclosure is to be considered as an exemplification of the principlesof the invention and is not intended to limit the invention to theembodiments illustrated.

In a first embodiment of the invention, the chondroitin sulfatesynthesis promoter contains, as an active ingredient, a gene encodingchondroitin sulfate glucuronyltransferase and/or a gene encodingchondroitin sulfate N-acetylgalactosaminyltransferase-1.

Examples of the gene coding for chondroitin sulfateglucuronyltransferase (CSGlcAT) include a nucleic acid moleculeincluding a nucleotide sequence defined by SEQ ID NO: 1 and encodinghuman CSGlcAT. The nucleic acid molecule may be obtained through, forexample, isolating RNA from human bone culture cells or similar cellsand performing RT-PCR by use of a primer design based on SEQ ID NO: 1.

The gene encoding CSGlcAT is not limited to a nucleic acid including anucleotide sequence defined by SEQ ID NO: 1. So long as the nucleic acidmolecule encodes a protein having CSGlcAT activity, there may beemployed a nucleic acid molecule which can be hybridized with acomplement of a nucleic acid of SEQ ID NO: 1 under stringent conditions.The stringent conditions include routine washing conditions employed insouthern blotting; e.g., 65° C., 0.1×SSC, 0.1% SDS solution.

So long as the nucleic acid molecule encodes a protein having CSGlcATactivity, the gene encoding CSGlcAT may be a nucleic acid moleculeincluding a nucleotide sequence identical to that defined by SEQ ID NO:1, except that one or more nucleotide(s) have been substituted, deleted,inserted, or transposed. The number of such nucleotides is preferably 2to 20, more preferably 2 to 10, particularly preferably 2 to 5.

The gene encoding CSGlcAT also includes a nucleic acid which has a highsequence identity (including as preferable examples, 75-100%, 80-100%,or 90-100% sequence identity, or 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.5%, or 99.9% sequence identity; higher sequence identities aremore preferable among the above examples) to a nucleotide sequencedefined by SEQ ID NO: 1.

The aforementioned homologue genes include naturally occurringspecies-dependent variants and variants produced through treatment suchas UV irradiation or treatment with a variation-inducing agent. The geneencoding CSGlcAT may be a gene derived from a non-human organism.However, when the product is employed as a drug for human use, ahuman-derived gene is preferred. Thus, the gene encoding CSGlcAT may bea nucleic acid molecule including a nucleotide sequence encoding anamino acid sequence defined by SEQ ID NO: 2.

As used herein, the term “CSGlcAT activity” refers to activity oftransferring glucuronate from a glucuronate donor to a non-reducing endN-acetylgalactosamine residue present in the chondroitin skeleton. Theactivity may be determined through a method disclosed in J. Biol. Chem.Oct. 11, 2002; 277 (41): 38179-88.

Examples of the gene encoding chondroitin sulfateN-acetylgalactosaminyltransferase-1 (CSGalNAcT-1) include a nucleic acidmolecule including a nucleotide sequence defined by SEQ ID NO: 3 andencoding human CSGalNAcT-1. The nucleic acid molecule may be obtainedthrough, for example, isolating RNA from human bone culture cells orsimilar cells and performing RT-PCR by use of a primer design on thebasis of SEQ ID NO: 3.

The gene encoding CSGalNAcT-1 is not limited to a nucleic acid includinga nucleotide sequence defined by SEQ ID NO: 3. So long as the nucleicacid molecule encodes a protein exhibiting CSGalNAcT-1 activity, anucleic acid molecule which can be hybridized with a complement of anucleic acid of SEQ ID NO: 3 under stringent conditions may be employed.The stringent conditions include routine washing conditions employed insouthern blotting; e.g., 65° C., 0.1×SSC, 0.1% SDS solution.

So long as the nucleic acid molecule encodes a protein exhibitingCSGalNAcT-1 activity, the gene encoding CSGalNAcT-1 may be a nucleicacid molecule including a nucleotide sequence identical to that definedby SEQ ID NO: 3, except that one or more nucleotide(s) have beensubstituted, deleted, inserted, or transposed. The number of suchnucleotides is preferably 2 to 20, more preferably 2 to 10, particularlypreferably 2 to 5.

The gene encoding CSGalNAcT-1 also includes a nucleic acid which has ahigh sequence identity (including as preferable examples, 75-100%,80-100%, or 90-100% sequence identity, or 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, 99.5%, or 99.9% sequence identity; higher sequenceidentities are more preferable among the above examples) to a nucleotidesequence defined by SEQ ID NO: 3.

The aforementioned homologue genes include naturally occurringspecies-dependent variants and variants produced through treatment suchas UV irradiation or treatment with a variation-inducing agent. The geneencoding CSGalNAcT-1 may be a gene derived from a non-human organism.However, when the product is employed as a drug for human use, ahuman-derived gene is preferred. Thus, the gene encoding CSGalNAcT-1 maybe a nucleic acid molecule including a nucleotide sequence encoding anamino acid sequence defined by SEQ ID NO: 4.

As used herein, the term “CSGalNAcT-1 activity” refers to activity oftransferring an N-acetylgalactosamine residue from anN-acetylgalactosamine donor to a non-reducing end D-glucuronate residueof an N-acetylgalactosamine acceptor substrate containing a sugar chainrepresented by formula:

GlcUA-Gal-Gal-Xyl

(wherein GlcUA represents a D-glucuronate residue, Gal represents aD-galactose residue, Xyl represents a D-xylose residue, and “-”represents a glycosidic linkage).

The CSGalNAcT-1 activity may be determined through a method disclosed inJ. Biol. Chem. Mar. 15, 2002; 277 (11): 8841-6.

When the aforementioned gene encoding chondroitin sulfateglucuronyltransferase and/or gene encoding chondroitin sulfateN-acetylgalactosaminyltransferase-1 is formulated as an activeingredient of the chondroitin sulfate synthesis promoter, the gene(s)may be formulated through a method for preparing a genetic druggenerally employed in a genetic therapy.

Preferably, the gene encoding chondroitin sulfate glucuronyltransferaseand a gene encoding chondroitin sulfateN-acetylgalactosaminyltransferase-1 are incorporated into an expressionvector.

In the above expression vector, regions relating to the gene expression(promoter region, enhancer region, operator region, etc.) may beappropriately arranged so that the gene is expressed. For example, theexpression vector may include vectors such as pcDNA3.1 (+), pCDM8, orpcDNA3 expression vector (product of Invitrogen).

The gene can be incorporated into an expression vector using techniqueswell known to the skilled artisan.

Preferably, the gene encoding chondroitin sulfate glucuronyltransferaseand a gene encoding chondroitin sulfateN-acetylgalactosaminyltransferase-1 are incorporated into a host cell.

Suitable host cells include a wide variety of prokaryotic and eukaryotichost cells. For example, the polypeptides of the invention may beexpressed in bacterial cells such as E. coli, insect cells (usingbaculovirus), yeast cells, fungal cells, insect cells, and plant oranimal cells, in particular mammalian cells. Other suitable host cellscan be found in Goeddel (1991) Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. A method forincorporating the gene into the host cell is not particularly limited.Methods for introducing exogenous genetic material into host cells arewell known to the skilled artisan, and the gene can be incorporated intothe host cell using such techniques. Preferably, a gene which isincorporated into an expression vector may be incorporated into the hostcell.

Specific examples of methods for incorporating the gene into the vector,and introducing genes to target tissue are shown below.

For example, the CSGlcAT gene or the CSGalNAcT-1 gene is ligated to apromoter which can be expressed in target tissue, to thereby produce agene construct, and the gene construct per se or a vector containing thegene construct is introduced into target tissue. Although the promotermay be a constitutionally expressed promoter such as a CMV promoter, thepromoter is preferably a tissue-specific promoter. Examples of thetarget tissue include articular cartilage and intervertebral disktissue. In the case where the target is chondrocytes or vertebral pulpcells, a promoter which functions in chondrocytes or vertebral pulpcells; e.g., a collagen gene promoter, may be employed. Examples ofemployable vectors include virus vectors such as an adenovirus vectorand a retrovirus vector, and plasmid vectors. In one possible mode, aCSGlcAT gene or a CSGalNAcT-1 gene is ligated to a type-2 collagenpromoter, to thereby form a gene construct, and the gene construct isintroduced to an adenovirus vector. Through incorporation of theproduct, the gene of the present invention can be introduced to targettissue (e.g., intervertebral disk tissue).

Other than the above gene construct, a nucleotide sequence defined bySEQ ID NO: 1 or a nucleotide sequence defined by SEQ ID NO: 3 may beligated with a tag sequence generally employed in a recombinationtechnique, to thereby form a gene construct. For example, a geneconstruct in which a V5 epitope tag is ligated to the nucleotidesequence may be produced. Of course, a gene construct in which both of apromoter and a tag sequence are ligated to the nucleotide sequence maybe produced. Through employment of a general recombination technique, agene construct in which a different sequence is ligated to thenucleotide sequence is expressed, whereby a fusion protein can beexpressed.

The gene construct containing a CSGlcAT gene or a CSGalNAcT-1 gene orthe vector containing the gene construct may be introduced incombination with a pharmacologically acceptable carrier. Examples of thepharmacologically acceptable carrier include physiological saline, astabilizer, a nuclease inhibitor, and a complexing agent such as EDTA.Alternatively, the gene construct containing a CSGlcAT gene or aCSGalNAcT-1 or the vector containing the gene construct may beintroduced in the form of a complex with ribosome. Still alternatively,cells such as chondrocytes transfected with the gene constructcontaining a CSGlcAT gene or a CSGalNAcT-1 gene or the vector containingthe gene construct may serve as a chondroitin sulfate synthesis promoterand be introduced to tissue such as cartilage.

The CSGlcAT gene or the CSGalNAcT-1 gene of the present invention may beapplied to a genetic therapy employing a gene gun (e.g., a gene gun or aparticle gun). The gene gun is a device for introducing an extraneousgene into a cell or tissue preferably through implanting a carrierharboring an extraneous gene or a composition containing an extraneousgene by pressure or other means. In other words, the present inventionmay employ a gene gun for introducing the gene of the present inventionthrough implanting a gene introduction carrier harboring the gene of thepresent invention into cartilage, a vertebral pulp cell, etc., whichgene gun has a tube for accelerating a gas such as helium, a mechanismfor holding the gene introduction carrier harboring the gene, and othermembers and which has dimensions suitable for introduction of the geneto the target tissue. The carrier which is employed in the gene gunmethod preferably has little affect on cells and tissue. Although othercariiers can be used, one preferred example of a carrier is goldparticles.

In one possible mode, the CSGlcAT gene or the CSGalNAcT-1 gene of theinvention is chemically or physically adsorbed onto spermidine-coatedgold particles, to thereby provide a gene-harboring carrier, and thecarrier is implanted into the target tissue such as cartilage or cellsby means of a gene gun, whereby the gene of the present invention isintroduced to the target tissue or cells.

Although no particular limitation is imposed on the administrationmethod, the genetic drug is preferably administered topically to thetarget tissue. The dose of the genetic drug may be appropriatelyadjusted in accordance with the expression efficiency of a nucleic acidmolecule and gravity of the target disease.

In a second embodiment of the present invention, the chondroitin sulfatesynthesis promoter contains, as an active ingredient, a chondroitinsulfate glucuronyltransferase or chondroitin sulfateN-acetylgalactosaminyltransferase-1.

Examples of the CSGlcAT protein include human CSGlcAT having an aminoacid sequence defined by SEQ ID NO: 2. The protein may be obtainedthrough extraction from an organism, followed by purification, orthrough a genetic engineering technique. For example, the protein isobtained through introducing a gene having a nucleotide sequence asdefined by SEQ ID NO: 1 into E. coli, animal cells, or a non-humantransgenic animal, to thereby express a recombinant protein, followed bypurification. Examples of the vector for introducing the gene into E.coli include pET vector (product of Novagen) and pGEX vector (product ofAmersham Pharmacia), and examples of the vector for introducing the geneinto animal cells include pcDNA vector (product of Invitrogen).

The CSGlcAT protein is believed to readily undergo variation such assubstitution of amino acids depending on species. However, if an aminoacid is substituted by another amino acid having similar characteristics(conservative substitution) or a certain amino acid not essential forexerting transfer activity is deleted, CSGlcAT activity is believed notto be affected. Therefore, so long as the nucleic acid molecule encodesa protein exhibiting CSGlcAT activity, in an amino acid sequence definedby SEQ ID NO: 2, one or more amino acid(s) may be substituted, deleted,inserted, or transposed. The number of such nucleotides is preferably 2to 20, more preferably 2 to 10, particularly preferably 2 to 5.

The CSGlcAT protein may be derived from a non-human organism. However,when the product is employed as a drug for human use, a human-derivedprotein (this includes human-derived protein obtained by means ofgenetic engineering using a human-derived gene, for example) ispreferred.

Examples of the CSGalNAcT-1 protein include human CSGalNAcT-1 proteinhaving an amino acid sequence defined by SEQ ID NO: 4. The protein maybe obtained through extraction from an organism, followed bypurification, or through a genetic engineering method. For example, theprotein is obtained through introducing a gene having a nucleotidesequence as defined by SEQ ID NO: 3 into E. coli, animal cells, or anon-human transgenic animal, to thereby express a recombinant protein,followed by purification.

The CSGalNAcT-1 protein is believed to readily undergo variation such assubstitution of amino acids depending on species. However, if an aminoacid is substituted by another amino acid having similar characteristics(conservative substitution) or a certain amino acid not essential forexerting transfer activity is depleted, CSGalNAcT-1 activity is believednot to be affected. Therefore, so long as the nucleic acid moleculeencodes a protein exhibiting CSGalNAcT-1 activity, in an amino acidsequence defined by SEQ ID NO: 4, one or more amino acid(s) may besubstituted, deleted, inserted, or transposed. The number of suchnucleotides is preferably 2 to 20, more preferably 2 to 10, particularlypreferably 2 to 5.

The CSGalNAcT-1 protein may be derived from a non-human organism.However, when the product is employed as a drug for human use, ahuman-derived protein (this includes human-derived protein obtained bymeans of genetic engineering using a human-derived gene, for example) ispreferred.

It is generally known that enzymes exist which have glycochains. Also,it is possible to express enzymes which have glycochains through geneticengineering. A chondroitin sulfate glucuronyltransferase or chondroitinsulfate N-acetylgalactosaminyltransferase-1 in the present invention mayhave parts such as glycochains in addition to their protein.

The CSGlcAT protein or the CSGalNAcT-1 protein per se may be employed asa chondroitin sulfate synthesis promoter. Alternatively, the aboveprotein may be blended with a pharmacologically acceptable carrier, andthe mixture may be employed as the synthesis promoter. Examples of thecarrier include a vehicle, a stabilizer, a tonicity agent, a surfactant,and a buffer. No particular limitation is imposed on the drug form ofCSGlcAT protein or CSGalNAcT-1 protein, and examples include injection,ointment, cream, fomentation, and liniment.

Although no particular limitation is imposed on the administrationmethod, the protein drug is preferably administered topically to thetarget tissue. The dose of the protein drug may be appropriatelyadjusted in response to gravity of the target disease or other factors.

No particular limitation is imposed on the diseases and disorders towhich the chondroitin sulfate synthesis promoter of the presentinvention is applied, so long as they can be treated by increasing thechondroitin sulfate level. However, the chondroitin sulfate synthesispromoter is preferably employed for improving functions of articularcartilage and intervertebral disk tissue. For example, the chondroitinsulfate synthesis promoter may serve as a treatment agent for a decreasein cartilage; articular diseases such as osteoarthritis and traumaticarticular disorders; or discopathy such as intervertebral diskdegradation or herniated disk, which are caused by aging, disease,injury, etc.

The present inventors have elucidated that each of CSGlcAT andCSGalNAcT-1 promotes synthesis of chondroitin sulfate in chondroidcells. Therefore, a compound which increases expression of a CSGlcATgene or a CSGalNAcT-1 gene may be a candidate for a low-molecular drugfor promoting synthesis chondroitin sulfate. In one screening procedurefor the chondroitin sulfate synthesis promoter, each test compound isadded to cartilage culture cells, and the protein level or mRNA level inrelation to CSGlcAT or CSGalNAcT-1 is determined. Among test compounds,a compound which increases the protein level or mRNA level in relationto CSGlcAT or CSGalNAcT-1 is selected.

Alternatively, a CSGlcAT gene and/or a CSGalNAcT-1 gene is expressed invitro in cultured chondrocytes, to thereby form cells exhibitingenhanced chondroitin sulfate synthesis performance, which serve astransplant chondrocytes. The cultured chondrocytes are preferablyhuman-derived. By use of a virus vector or a plasmid vector containing aCSGlcAT gene and/or a CSGalNAcT-1 gene, these genes are expressed incultured chondrocytes, and promotion of chondroitin sulfate synthesis isconfirmed. Thereafter, the thus-gene-introduced cultured chondrocytesare transplanted into a disordered site.

Further the present invention provides a method for promotingchondroitin sulfate synthesis in a living subject wherein the livingsubject includes a joint, a cartilage, an intervertebral disc or a cell(including a chondrocyte), the method comprising the steps of:

providing a gene comprising a gene encoding chondroitin sulfateglucuronyltransferase or a gene encoding chondroitin sulfateN-acetylgalactosaminyltransferase-1; and

introducing the gene into the living subject.

Preferably the step of introducing the gene into the living subject isperformed with a gene gun or via injection. Please refer to the abovesaid explanations about the gene gun or gene therapy.

Preferably a living subject is a living subject derived from mammal suchas human, domestic animal (including for example, a horse, cattle, andpig), or pet (including for example, dog and cat), for example.

Further the present invention provides a method for promoting achondroitin sulfate synthesis in a living subject wherein the livingsubject includes a joint, a cartilage, an intervertebral disc or a cell(including a chondrocyte), the method comprising the steps of:

providing a chondroitin sulfate glucuronyltransferase or chondroitinsulfate N-acetylgalactosaminyltransferase-1; and

introducing a chondroitin sulfate glucuronyltransferase or chondroitinsulfate N-acetylgalactosaminyltransferase-1 into the living subject.

Preferably a living subject is a living subject derived from mammal suchas human, domestic animal (including for example, a horse, cattle, andpig), or pet (including for example, dog and cat), for example.

Preferbly the step of introducing a chondroitin sulfateglucuronyltransferase or chondroitin sulfateN-acetylgalactosaminyltransferase-1 into the living subject can beperformed via injection, for example.

Further the present invention provides a method for treating anarticular disease or discopathy, the method comprising the steps of:

applying a therapeutically effective amount of an active ingredientcomprising a chondroitin sulfate glucuronyltransferase, a gene encodingchondroitin sulfate glucuronyltransferase, a chondroitin sulfateN-acetylgalactosaminyltransferase-1, or a gene encoding a chondroitinsulfate N-acetylgalactosaminyltransferase-1 with the affected joint orintervertebral disc; and

promoting a chondroitin sulfate synthesis in the affected joint orintervertebral disc.

The method to apply the active ingredient with the affected joint orintervertebral disc is not particularly limited. When the activeingredient is selected from a gene encoding chondroitin sulfateglucuronyltransferase and a gene encoding a chondroitin sulfateN-acetylgalactosaminyltransferase-1, gene gun or injection can be usedpreferably. When the active ingredient is selected from a chondroitinsulfate glucuronyltransferase and a chondroitin sulfateN-acetylgalactosaminyltransferase-1, injection can be used preferably.

A therapeutically effective amount is a concentration or amount of theactive ingredient which is effective for achieving a therapeutic effect.This amount may also be determined empirically.

EXAMPLES

The present invention will next be described in detail by way ofexamples, which should not be construed as limiting the inventionthereto.

Example 1 Expression of Chondroitin Sulfate Glycosyltransferase inCartilage

Humeri were obtained from mouse E16.5 embryos, and in situ hybridizationwas performed in order to confirm expression of chondroitin sulfateglycosyltransferase in cartilage.

Digoxigenin (DIG)-11-UTP-labeled single-strand antisense RNA probescorresponding to mouse CSS-1, CSS-2, CSS-3, CSGlcAT, CSGalNAcT-1,CSGalNAcT-2, aggrecan, versican, and type-10 collagen α1 chain (Col10a1)were prepared by use of a DIG RNA labeling kit (product of BoehringerMannheim) according to the manufacturer's instructions. Each humerussample was collected from a mouse E16.5 embryo, and a paraffin sectionthereof was prepared. After removal of paraffin, the section was fixedwith 4% paraformaldehyde for 10 minutes and treated with 20-ng/mLprotein kinase K (product of Roche) at 37° C. for 7 minutes. Anotherfixation was performed with 4% paraformaldehyde for 10 minutes, followedby treating with 0.2-mol/L hydrochloric acid for 10 minutes and0.25-mol/L acetic anhydride/0.1-mol/L triethylamine (TEA) for 10minutes. The thus-treated section was dehydrated with ethanol and driedunder air. The dried section was hybridized with 1.0-μg/mL RNA probe at50° C. for 20 hours. The product was washed with 2×SSC (buffer:0.3-mol/L sodium citrate and 3-mol/L sodium chloride)/50% formamide.Subsequently, the washed product was treated with 20 μg/mL RNaseA/TNEbuffer (10-mmol/L Tris-HCl (pH: 8.0), 0.5-mol/L sodium chloride, and1-mmol/L dihydrogen sodium ethylenediaminetetraacetate (EDTA)) at 37° C.for 20 minutes, and washed with 2×SSC at 50° C. for 10 minutes and a20×SSC at 50° C. for 20 minutes. The product was blocked with a 1.5%blocking reagent (product of Applied Biosystems) at 37° C. for one hour,reacted with a 1:500 anti-DIG antibody (product of Roche) at 37° C. forone hour, and colored with an NBT/BIP (nitroblue tetrazoliumhydrochloride/bromochloroindolylphosphoric acid, product of Roche) atroom temperature for 56 hours. Hematoxylin staining and alcian bluestaining were also performed.

The results are shown in FIG. 1.

Among six chondroitin sulfate glycosyltransferases, considerableexpression of mRNA was confirmed in cartilage for fourglycosyltransferases: CSS-1, CSS-2, CSGlcAT, and CSGalNAcT-1 CSS-3 andCSGalNAcT-2 were not found to be expressed. In articular cartilagecovering the articular plate and growth cartilage, the above fourglycosyltransferases were colocalized with core proteins of aggrecan andversican, confirming expression of the four glycosyltransferases.

Example 2 Expression Patterns of Chondroitin SulfateGlycosyltransferases During Chondrocyte Differentiation

Chondrogenic ATDC5 cells were cultured in Dulbecco's modified Eaglemedium (DMEM)/F-12 medium containing 5% fetal bovine serum (FBS),penicillin and streptomycin. In order to induce differentiation ofchondrocytes, the cells at confluency were treated with 10-μg/mL bovineinsulin, 10-μg/mL human transferrin, and 3×10⁻⁸-mol/L sodium selenite.

On days 1, 3, 5, 7, 14, and 21 after induction of differentiation (day0: at about 80% confluency (growing phase) before induction ofdifferentiation), the expression amount of mRNA (a gene transcriptionproduct) corresponding to mouse CSS-1, CSS-2, CSS-3, CSGlcAT,CSGalNAcT-1, CSGalNAcT-2, or aggrecan was determined through real timeRT-PCR.

mRNA was extracted by use of Micro-FastTrack (product of Invitrogen),and cDNA was reverse transcripted therefrom by use of SuperScriptFirst-Strand (product of Invitrogen). The primers andfluorescence-labeled probes corresponding to the genes are shown inTable 1, and were designed by use of Primer Express 1.0 software.

TABLE 1 Gene (GenBank Accession No.) aggrecan For- ctgcccttgccccgtaaderived ward (SEQ ID NO: 5) from mouse primer (NM_007424) Re-gacaggtcaaagatgggctttg verse (SEQ ID NO: 6) primer Probeccctgggcagcgtgatcctcac (SEQ ID NO: 7) CSS-1 For- ctggacctgctgctcctgtatderived ward (SEQ ID NO: 8) from mouse primer (XM_194358) Re-tcttcagggaattggacaggaa verse (SEQ ID NO: 9) primer Probecagcagaccttcagcaagatgcagtttgt (SEQ ID NO: 10) CSS-2 For-gggctttggagtcttgctctct derived ward (SEQ ID NO: 11) from mouse primer(XM_129886) Re- ggcgagcactgacgatgtc verse (SEQ ID NO: 12) primer Probeacagcaactgcgcccccacct (SEQ ID NO: 13) CSS-3 For-ggaaactgggttttggagagacta derived ward (SEQ ID NO: 14) from mouse primer(XM_128873) Re- ccgtaagccagataggatgacttta verse (SEQ ID NO: 15) primerProbe acggaatccaatgcatttacaaaagcgatc (SEQ ID NO: 16) CSG1cAT For-tggccgtcgcggttaa derived ward (SEQ ID NO: 17) from mouse primer(NM_133913) Re- tccatgagacaccacctgcat verse (SEQ ID NO: 18) primer Probecgtacagtagcacatcacttccctcggttact (SEQ ID NO: 19) CSGa1NAcT-1 For-tgagctggtagaagccatcga derived ward (SEQ ID NO: 20) from mouse primer(BC057630) Re- gttcggtaaatcccttctatgaagtc verse (SEQ ID NO: 21) primerProbe cggccctggagagtctaaacagccct (SEQ ID NO: 22) CSGa1NAcT-2 For-gctgagccaggtaaaaaggtgtt derived ward (SEQ ID NO: 23) from mouse primer(NM_030165) Re- aaaccagagtcctttttatgaacca verse (SEQ ID NO: 24) primerProbe caggacgtgccgccccctg (SEQ ID NO: 25)

PCR was performed by use of 5-μL cDNA, 25-μL TaqMan Universal PCR MasterMix (product of Applied Biosystems), 100-nmol/L probe, and 100-nmol/Lprimer (total 50 μL), and determination was performed on a 96-well plateby means of an ABI Prism 7700 (product of Applied Biosystems).

The aforementioned mRNA levels were standardized by an amount of a genetranscription product (mRNA level) of glycerinaldehyde triphosphatedehydroganase (GAPDH) determined through the same method. Thethus-standardized mRNA expression levels were plotted as fold numbersagainst day 0 (before induction of differentiation (FIG. 2)).

Example 3 Expression Patterns of Chondroitin SulfateGlycosyltransferases During Chondrocyte Differentiation

Chondrogenic N1511 cells were cultured in a minimum essential medium α(α-MEM medium) supplemented with 10% FBS, penicillin, and streptomycinat 37° C. under 5% CO2. The cells at confluency were treated with1×10⁻⁶-mol/L dexamethasone solution and 1×10⁻⁷-mol/L rat parathyroidhormone (PTH) solution, to thereby induce differentiation. In a mannersimilar to that of Example 2, on days 1, 3, 5, 7, 14, and 21 afterinduction of differentiation (day 0: at about 80% confluency (growingphase) before induction of differentiation), the level of mRNA (a genetranscription product)corresponding to CSS-1, CSS-2, CSS-3, CSGlcAT,CSGalNAcT-1, CSGalNAcT-2, or aggrecan was determined. In a mannersimilarly to that of Example 2, the aforementioned mRNA levels werestandardized by an mRNA level of GAPDH. The thus-standardized mRNAexpression levels were plotted as fold numbers with respect to those atday 0 (before induction of differentiation) (FIG. 3).

As is clear from FIGS. 2 and 3, in both the case where differentiationof ATDC5 cells was induced and the case where differentiation of N1511cells was induced, CSGlcAT transcriptional level and CSGalNAcT-1transcriptional level increased remarkably, as compared with otherenzymes. Meanwhile, it has already been known that aggrecan synthesis ispromoted in chondrogenic cells such as ATDC5 cells and N1511 cellsduring an induced differentiation step. For example, in the case ofATDC5 cells, the aggrecan core protein transcription product (mRNA)level reaches the peak 7 to 10 days after induction of differentiation.In the above experiments, the aggrecan transcriptional level reached thepeak approximately at day 7, indicating that transcriptional levels ofCSGlcAT and CSGalNAcT-1 are correlated to the change in genetranscriptional level with respect to aggrecan core protein. Therefore,CSGlcAT and CSGalNAcT-1 are believed to be involved in chondroitinsulfate chain synthesis in cartilage. Note that a CSS-3 transcriptionproduct (mRNA) was not detected over the period of the experiment.

Example 4 Expression Patterns of Chondroitin SulfateGlycosyltransferases in Mouse Embryonic Fibroblasts

Mouse embryonic fibroblasts (MEFs, product of BD Bioscience) werecultured in a DMEM medium supplemented with 10% FBS, penicillin, andstreptomycin at 37° C. under 5% CO2.

At a growing phase (about 80% confluency) and one week after confluency,the level of mRNA (a gene transcription product)corresponding to CSS-1,CSS-2, CSGlcAT, CSGalNAcT-1, CSGalNAcT-2, or aggrecan was determinedthrough the same method as employed in Example 2. Similar to Example 2,the mRNA levels were standardized by an mRNA level of GAPDH.

Expression levels (mRNA levels) of the genes at the growing phase (day0) before induction of differentiation and at one week after inductionof differentiation in Examples 2 and 3, and expression levels (mRNAlevels) of the genes at the growing phase and at one week afterconfluency in Example 4 were plotted as fold numbers with respect tothose at the growing phase of ATDC5 cells determined in Example 2 (FIG.4).

mRNA expression levels of CSGlcAT, CSGalNAcT-1, and aggrecan wereremarkably increased one week after induction of differentiation inchondrogenic cells such as ATDC5 cells and N1511 cells, and the increasewas found to be correlated with increase in aggrecan. In contrast, mRNAexpression level of CSGlcAT was virtually unobserved in mouse embryonicfibroblasts (MEFs), which are not differentiated to chondrocytes, andmRNA expression level of CSGalNAcT-1 was not considerably changed ascompared with chondrogenic cells.

In Examples 2, 3, and 4, expression of CSGlcAT and CSGalNAcT-1correlated highly with chondrocyte differentiation in chondrogeniccells.

Similar to Example 1, mRNA expression level of CSGalNAcT-2 was greaterin fibroblasts and was virtually unobserved in chondrogenic cells.

Example 5 Establishment of CSGlcAT-Transfected Cells andCSGalNAcT-1-Transfected Cells

Rat chondrosarcoma cells (LTC cells) were transfected with human CSGlcATand CSGalNAcT-1 expression vectors, respectively, followed by culturing.The transfected cells were selected by use of 1-mg/mL G418 disulfate(product of Nacalai Tesque).

Each of the above expression vectors was produced by incorporating cDNAof human CSGlcAT or cDNA of CSGalNAcT-1 into a pcDNA3.1 (+) vector(product of Invitrogen) (the expression vectors were donated by Dr. Gotoof Advanced Industrial Science and Technology, Research Center forGlycoscience).

Rat chondrosarcoma cells (LTC cells) were cultured in a DMEM mediumsupplemented with 10% FBS, penicillin, and streptomycin at 37° C. under5% CO2. Each expression vector was cut with a restriction enzyme PvuI(product of New England Biolabs) and linearized, and the LTC cells weretransfected with the vector using FuGENE6 (product of Roche) inaccordance with the manufacturer's instructions. On the day followingtransfection, 1-mg/mL G418 was added to the above culture, and culturingwas further performed for 10 days, whereby transfected cells wereselected. As a control, Mock-transfected cells were established in asimilar manner through transfection with pcDNA3.1 (+) (product ofInvitrogen) instead of the above enzymes.

Example 6 Analysis of Transfected Cells

The transfected cells established in Example 5 were analyzed as follows.

(1) Determination of Expression Levels of Enzymes:

CSGlcAT, CSGalNAcT-1, and Mock were analyzed in terms of expressionlevels of the corresponding exogenous enzymes (human-derived) andendogenous enzymes (rat-derived) according to the method employed inExample 2 through real time RT-PCR. The primers and fluorescence-labeledprobes corresponding to the genes are shown in Table 2, and weredesigned by use of Primer Express 1.0 software.

TABLE 2 Gene (GenBank Accession No.) CSG1cAT For-ctagaccaaagtgatgaagacttcaaac derived ward (SEQ ID NO: 26) from humanprimer (AB037823) Re- tgtaccgagtcctgagcacctt verse (SEQ ID NO: 27)primer Probe ctacagggaccccaacaagccctacaag (SEQ ID NO: 28) CSGa1NAcT-1For- agcagcaccgcaactacgt derived ward (SEQ ID NO: 29) from human primer(AB081516) Re- ctggcttggtactgcccatt verse (SEQ ID NO: 30) primer Probectgaagcggcagatcgcacagct (SEQ ID NO: 31) CSG1cAT For- tggccgtcgctgttaaderived ward (SEQ ID NO: 32) from rat primer (XM_216063) Re-tccatgagacaccacctgcat verse (SEQ ID NO: 33) primer Probecgtacagtggcacatcacttccctcggttact (SEQ ID NO: 34) CSGa1NAcT-1 For-tgagctagtggaagctatcga derived ward (SEQ ID NO: 35) from rat primer(XM_224757) Re- gttcggtagatcccttctatgaagtc verse (SEQ ID NO: 36) primerProbe cagccctggagagtctaaacagccct (SEQ ID NO: 37)

Reference to the respective expression vectors as controls, copy numbers(transcriptional levels) of human CSGlcAT and CSGalNAcT-1 and ratCSGlcAT and CSGalNAcT-1 are shown in FIG. 5.

(2) Alcian Blue Staining and Immunostaining:

In Alcian Blue staining, each transfected cell was seeded onto a DMEMmedium supplemented with 10% FBS, penicillin, and streptomycin placed ina 6-well plate, followed by culturing at 37° C. under 5% CO₂. One dayafter, the culture was fixed in an 80%-confluency state with 4%paraformaldehyde and treated for 30 minutes with 0.1% Alcian Bluedissolved in 0.1-mol/L hydrochloric acid (FIG. 6, upper).

Immunostaining was performed by use of a mouse monoclonal antibodyreacting with chondroitin-4-sulfate (C4S)(Ab-Chondroitin-4-Sulfate/chicken (LY111), product of SEIKAGAKUCORPORATION) and a rabbit polyclonal antibody against aggrecan (FIG. 6,lower) (the rabbit polyclonal antibody against aggrecan was donated byDr. Yada of Institute for Molecular Science of Medicine, Aichi MedicalUniversity).

(3) Determination of Chondroitin Sulfate Synthesis Activity:

LTC cells transfected with a CSGlcAT gene and those transfected with aCSGalNAcT-1 gene were cultured in cell culture dishes (3003, product ofFalcon) containing a DMEM medium supplemented with 10% FBS, penicillin,and streptomycin, at 37° C. under 5% CO2. On the following day,100-pCi/mL³ ⁵S sulfate was added to each culture, followed by culturingfor 24 hours, so as to label the cells. Subsequently, cells and aculture medium were separately collected and extracted with 0.1-mol/Lsodium hydride solution at 4° C. for 16 hours. Subsequently, the systemwas neutralized with 4-mol/L acetic acid. Under 20-mmol/L Tris-HCl (pH:8.0) buffered conditions, 5-μL protein kinase K (20 mg/mL, product ofRoche) was added to the system, followed by treatment at 37° C. for 12hours. Through heating to 100° C., protein kinase K was inactivated.Then, 10-μL DNaseI (10 mg/mL, product of Boehringer Mannheim) and 10-μLRNaseA (10 mg/mL, product of Wako) were added to the above system,followed by treatment at 37° C. for two hours. Glycosaminoglycan (GAG)chains were collected from the reaction mixture. The thus-collected GAGchains were applied to a DEAE-Sepharose column. The column was washedwith a 0.2-mol/L aqueous sodium chloride solution, and GAG fractionswere eluted with an aqueous 2-mol/L sodium chloride solution. Thethus-obtained GAG was precipitated with a 1.3% aqueous potassium acetatesolution and 95% ethanol, and the thus-obtained precipitate wassuspended in a buffer (50-mmol/L Tris-HCl (pH: 7.5)−0.2-mol/L sodiumchloride). The GAG suspension was divided into three portions, and theportions were treated with a heparitinase mixture containingheparitinase I, heparitinase II, and heparinase (all enzymes areproducts of SEIKAGAKU CORPORATION); treated with a chondroitinase ABC(product of SEIKAGAKU CORPORATION); and non-treated, respectively.Subsequently, each of the thus-treated GAG samples was applied to aSuperrose 6 (product of Amersham Biosciences) column which had beenequilibrated with an aqueous solution (50-mmol/L Tris-HCl (pH:7.5)−0.2-mol/L sodium chloride), and eluted with the aqueous solution(50-mmol/L Tris-HCl (pH: 7.5)−0.2-mol/L sodium chloride), whereby 0.5-mLfractions were collected. Radioactivity of labeled GAG contained in eachfraction was measured by means of a liquid scintillation counter, and anelution curve was obtained through the data of scintillation counting(FIG. 7, lower).

The graph in FIG. 7 (upper) shows scintillation counts (transfected celllysate and culture medium) of digested chondroitin sulfate fractions(19.5 mL to 21 mL) obtained from chondroitinase ABC-treated samples(lower graphs in elution curve graphs). Each scintillation count is asum of the count attributed to the respective transfected cells and thatattributed to the respective culture medium.

(4) Disaccharide Analysis of Chondroitinase-ABC Digests of GAG Samples:

With reference to a method disclosed in Analytical Biochemistry, 177,327-332 (1989), chondroitinase-ABC-digested products ofglycosaminoglycan chains synthesized by the transfected cells wereanalyzed in terms of disaccharide composition.

LTC cells transfected with a CSGlcAT gene and those transfected with aCSGalNAcT-1 gene were cultured in cell culture dishes (3003, product ofFalcon) containing a DMEM medium supplemented with 10% FBS, penicillin,and streptomycin, at 37° C. under 5% CO2 for 24 hours. Subsequently,cells and a culture medium were separately collected and extracted0.1-mol/L sodium hydride solution at 4° C. for 16 hours. Subsequently,the system was neutralized with 4-mol/L acetic acid. Under 20-mmol/LTris-HCl (pH: 8.0) buffered conditions, 5-μL protein kinase K (20 mg/mL,product of Roche) was added to the system, followed by treating at 37°C. for 12 hours. Through heating to 100° C., protein kinase K wasinactivated. Then, 10-μL DNaseI (10 mg/mL, product of BoehringerMannheim) and 10-μL RNaseA (10 mg/mL, product of Wako) were added to theabove system, followed by treating at 37° C. for two hours.Glycosaminoglycan (GAG) chains were collected from the reaction mixture.The thus-collected GAG chains were applied to a DEAE-Sepharose column.The column was washed with a 0.2-mol/L aqueous sodium chloride solution,and GAG fractions were eluted with an aqueous 2-mol/L sodium chloridesolution. The thus-obtained GAG was precipitated with a 1.3% aqueouspotassium acetate solution and 95% ethanol, and the thus-obtainedprecipitate was suspended in a buffer (50-mmol/L Tris-HCl (pH:7.5)−0.2-mol/L sodium chloride). The GAG suspension was treated with achondroitinase ABC (product of SEIKAGAKU CORPORATION).

GAG-digested products formed through chondroitinase ABC treatment wereanalyzed through fluorometric postcolumn HPLC so as to confirm presenceof disaccharide (FIG. 8).

Each of the graphs in FIG. 8 (upper) shows total peak area of therespective sugar with respect to Mock, CSGlcAT, and CSGalNAcT-1 obtainedin disaccharide analysis (see FIG. 8. lower). Each bar shows the sum ofpeak areas (0S, 4S, and Di(4,6)S). The values were standardized on thebasis of a peak area obtained from a disaccharide standard sample (10μmol/L) through the same method, and were reduced to amount of GAG/dish.

As used herein, “Di(4,6)S” denotes an unsaturated disaccharide whichforms a disaccharide unit of chondroitin sulfate and which has a4-sulfate group and 6-sulfate group (instead of 4-hydroxyl group and6-hydroxyl group) in N-acetylgalactosamine; “4S” denotes an unsaturateddisaccharide which forms the disaccharide unit and which has only a4-sulfate group in N-acetylgalactosamine; and “0S” denotes anunsaturated disaccharide having no sulfate unit in the disaccharideunit.

In analysis (1) above, CSGlcAT-transfected cells andCSGalNAcT-1-transfected cells exhibited a remarkably high expression ofthe introduced genes, as compared with the control.

In immunostaining analysis (2) above, as compared with the control(Mock), CSGlcAT-transfected cells and CSGalNAcT-1-transfected cells havehigher chondroitin 4-sulfate contents, and have an aggrecan core proteincontent almost equivalent to that of the control. The LTC cells arematuration chondrocytes, and cell characteristics remain duringcontinuous culturing. In other words, the LTC cells constantlysynthesize predetermined aggrecan molecules. Consequently, the coreprotein amount of aggrecan was found to be constant.

In analysis (3) above, transfected cells overexpressing CSGlcAT andCSGalNAcT-1 exhibited chondroitin sulfate synthesis activity of 1.6 foldand 2.2 fold, respectively, as compared with the control. As shown inthe graph in FIG. 7 (middle, right), chondroitin sulfate elution curvesof Mock, CSGlcAT, and CSGalNAcT-1-transfected cells, which had beentreated with the heparitinase mixture, exhibited a common peak profilecorresponding to fractions. Therefore, the synthesized chondroitinsulfate chains released to the culture medium had no variation in sizeamong samples.

The above analyses revealed that, in CSGlcAT-transfected cells andCSGalNAcT-1-transfected cells, the amount of aggrecan core protein wasnot changed, but chondroitin sulfate chains of the same length weresynthesized 1.6 fold and 2.2 fold in amount, respectively, indicatingthat the number of chondroitin sulfate chains in one aggrecan moleculeincreased 1.6 fold and 2.2 fold, respectively.

In analysis (4), chondroitin sulfate synthesized in each transfectedcells exhibited a large amount of disaccharide essential units having a4-sulfate structure. As compared with CSGlcAT-transfected cells,CSGalNAcT-1-transfected cells synthesized chondroitin sulfate in alarger amount.

Examples 2, 3, and 4 have revealed that, among chondroitin sulfateglycosyltransferases believed to be related to biosynthesis ofchondroitin sulfate, CSGlcAT and CSGalNAcT-1 are closely related tobiosynthesis of chondroitin sulfate in cartilage. In particular,CSGalNAcT-1 is closely related to the biosynthesis. The Examples havealso revealed that an increase in expression levels of CSGlcAT andCSGalNAcT-1 leads to an increase in amounts of chondroitin sulfatechains bound to the core protein of one aggrecan molecule, as comparedwith the control.

Accordingly, when expression or activity of these enzymes is promoted,or these enzymes are exogenously administered, synthesis of chondroitinsulfate chains is expected to be promoted in cartilage, whereby thenumber of chondroitin sulfate chains which forms parts of aggrecan canbe increased.

Example 7 Biosynthesis of Chondroitin Sulfate (CS) in MouseIntervertebral Discs by in vivo Gene Delivery of CSGalNAcT-1

cDNA encoding hCSGalNAcT-1 (CSGalNAcT-1 of human origin) was prepared byPCR using primers (5′-CACCATGATGATGGTTCGCCG-3′,5′-TGTTTTTTTGCTACTTGTCTTCTG-3′) and a plasmid (hCSGalNAcT-1/pcDNA3.1) asa template, and the prepared cDNA was subcloned into adenoviralexpression vectors (pAd/CMV/V5-DEST, manufactured by Invitrogen, Co.)through entry-vectors (pENT/D-TOPO, manufactured by Invitrogen, Co.) toobtain adenoviral vectors for expression of hCSGalNAcT-1. Next, theadenoviral vectors for expression of hCSGalNAcT-1 were transduced into293A cells (manufactured by Invitrogen, Co.) using an ViraPowerAdenovirus Expression System (manufactured by Invitrogen, Co.) accordingto the manufacturer's instructions to obtain adenoviral particles forexpression of hCSGalNAcT-1.

The particles' titer was checked according to the above manufacturer'sinstructions. 1.5×107 plaque-forming unit (pfu) ADENOVIRAL particleswere dissolved in 30 μl phosphate buffer saline and injected into mouseintervertebral discs of 4 month old ICR mice at a dosage of 1.5×107plaque-forming unit (pfu). In addition, adenoviral particles wereprepared with pAd/CMV/V5-GW/Lac-Z (manufactured by Invitroegn, Co.)according to the above-described same procedures and used as a negativecontrol (“mock”).

At day 7 after the injection the mice were dissected and theintervertebral discs were stained with alcian blue. Quantitative imagingby NIH-image (produced by National Institute of Health) were thenperformed on the results of alcian blue stain and the alcian blue stainwere figured in quantitative data (FIG. 10).

Histological analysis showed intense alcian blue staining in thepericellular zone of the nucleus pulposus cells in the disc andchondrocytes in the vertebral endplate of the mice injected with theCSGalNAcT-1 gene, compared to that of mice injected with the mock (FIG.9). This result teaches that the volume or biosynthesis of CS in mouseintervertebral discs is increased by in vivo gene delivery ofCSGalNAcT-1.

And upon the above, it is asserted that genetic therapy with delivery ofthe gene of CSGalNAcT-1 into a joint increases the volume of CS in thatjoint toward attacking and/or curing diseases or symptoms associatedwith the decrease of the volume of CS.

Results in the present invention teach that the introduction ofCSGalNAcT-1 gene and/or CSGlcAT gene, or exogenous administration ofCSGalNAcT-1 protein and/or CSGlcAT protein promotes expression oractivity of these enzymes in joints such as cartilage or intervertebraldisc. Then the synthesis of chondroitin sulfate chain can be promoted,resulting in an increase of chondroitin sulfate chains of aggrecan.

In addition, it is believed that improved water retention of aggrecan byvirtue of an increase in chondroitin sulfate chains, improves functionsof cartilage and imparts an increase in shock absorbing and frictionreducing (lubrication) performance there to. When expression or activityof these enzymes is promoted, or these enzymes are exogenouslyadministered, formation of cartilage is expected to be promoted. Forexample, a cartilage formation promoter and a cartilage repairing agentfor repairing damaged cartilage can be provided. Through employment of adrug or drug treatment comprising CSGalNAcT-1 or CSGlcAT as an activeingredient, or a genetic therapy employing a gene encoding CSGalNAcT-1or CSGlcAT, a cartilage repairing agent or a treatment agent forarticular disease and discopathy can be effectively provided.

1. A chondroitin sulfate synthesis promoter comprising, as an activeingredient, at least one of a gene encoding chondroitin sulfateglucuronyltransferase and a gene encoding chondroitin sulfateN-acetylgalactosaminyltransferase-1.
 2. A chondroitin sulfate synthesispromoter as described in claim 1, wherein the gene encoding chondroitinsulfate glucuronyltransferase is a nucleic acid molecule including anucleotide sequence defined by SEQ ID NO:
 1. 3. A chondroitin sulfatesynthesis promoter as described in claim 1, wherein the gene encodingchondroitin sulfate glucuronyltransferase is a nucleic acid moleculewhich can be hybridized with a complement of a nucleic acid of SEQ IDNO: 1 under stringent conditions.
 4. A chondroitin sulfate synthesispromoter as described in claim 1, wherein the gene encoding chondroitinsulfate glucuronyltransferase is a nucleic acid molecule including anucleotide sequence identical to that defined by SEQ ID NO: 1, exceptthat one or more nucleotide(s) have been substituted, deleted, inserted,or transposed, wherein the nucleic acid molecule encodes a proteinhaving activity of transferring glucuronate from a glucuronate donor toa non-reducing end N-acetylgalactosamine residue present in thechondroitin skeleton.
 5. A chondroitin sulfate synthesis promoter asdescribed in claim 1, wherein the gene encoding chondroitin sulfateglucuronyltransferase is a nucleic acid molecule including a nucleotidesequence encoding an amino acid sequence defined by SEQ ID NO:
 2. 6. Achondroitin sulfate synthesis promoter as described in claim 1, whereinthe gene encoding chondroitin sulfateN-acetylgalactosaminyltransferase-1 is a nucleic acid molecule includinga nucleotide sequence defined by SEQ ID NO:
 3. 7. A chondroitin sulfatesynthesis promoter as described in claim 1, wherein the gene encodingchondroitin sulfate N-acetylgalactosaminyltransferase-1 is a nucleicacid molecule which can be hybridized with a complement of a nucleicacid of SEQ ID NO: 3 under stringent conditions.
 8. A chondroitinsulfate synthesis promoter as described in claim 1, wherein the geneencoding chondroitin sulfate N-acetylgalactosaminyltransferase-1 is anucleic acid molecule including a nucleotide sequence identical to thatdefined by SEQ ID NO: 3, except that one or more nucleotide(s) have beensubstituted, deleted, inserted, or transposed, wherein the nucleic acidmolecule encodes a protein having activity of transferring anN-acetylgalactosamine residue from an N-acetylgalactosamine donor to anon-reducing end D-glucuronate residue of an N-acetylgalactosamineacceptor substrate containing a sugar chain represented by formula;GlcUA-Gal-Gal-Xyl (wherein GlcUA represents a D-glucuronate residue, Galrepresents a D-galactose residue, Xyl represents a D-xylose residue, and“-” represents a glycosidic linkage).
 9. A chondroitin sulfate synthesispromoter as described in claim 1, wherein the gene encoding chondroitinsulfate N-acetylgalactosaminyltransferase-1 is a nucleic acid moleculeincluding a nucleotide sequence encoding an amino acid sequence definedby SEQ ID NO:
 4. 10. A chondroitin sulfate synthesis promoter asdescribed in claim 1, wherein at least one of a gene encodingchondroitin sulfate glucuronyltransferase and a gene encodingchondroitin sulfate N-acetylgalactosaminyltransferase-1 are incorporatedinto an expression vector.
 11. A chondroitin sulfate synthesis promoteras described in claim 1, wherein at least one of a gene encodingchondroitin sulfate glucuronyltransferase and a gene encodingchondroitin sulfate N-acetylgalactosaminyltransferase-1 are incorporatedinto a host cell.
 12. A chondroitin sulfate synthesis promotercomprising, as an active ingredient, at least one of a chondroitinsulfate glucuronyltransferase and a chondroitin sulfateN-acetylgalactosaminyltransferase-1.
 13. A chondroitin sulfate synthesispromoter as described in claim 12, wherein the chondroitin sulfateglucuronyltransferase is an enzyme comprising a protein which includesan amino acid sequence defined by SEQ ID NO:
 2. 14. A chondroitinsulfate synthesis promoter as described in claim 12, wherein thechondroitin sulfate glucuronyltransferase is an enzyme comprising aprotein which includes an amino acid sequence identical to that definedby SEQ ID NO: 2, except that one or more amino acid residue(s) have beensubstituted, deleted, inserted, or transposed, wherein the protein hasactivity of transferring glucuronate from a glucuronate donor to anon-reducing end N-acetylgalactosamine residue present in thechondroitin skeleton.
 15. A chondroitin sulfate synthesis promoter asdescribed in claim 12, wherein the chondroitin sulfateN-acetylgalactosaminyltransferase-1 is an enzyme comprising a proteinwhich includes an amino acid sequence defined by SEQ ID NO:
 4. 16. Achondroitin sulfate synthesis promoter as described in claim 12, whereinthe chondroitin sulfate N-acetylgalactosaminyltransferase-1 is an enzymecomprising a protein which includes an amino acid sequence identical tothat defined by SEQ ID NO: 4, except that one or more amino acidresidue(s)have been substituted, deleted, inserted, or transposed,wherein the protein has activity of transferring anN-acetylgalactosamine residue from an N-acetylgalactosamine donor to anon-reducing end D-glucuronate residue of an N-acetylgalactosamineacceptor substrate containing a sugar chain represented by formula;GlcUA-Gal-Gal-Xyl (wherein GlcUA represents a D-glucuronate residue, Galrepresents a D-galactose residue, Xyl represents a D-xylose residue, and“-” represents a glycosidic linkage).
 17. A treatment agent for at leastone of an articular disease and discopathy comprising, as an activeingredient, a chondroitin sulfate synthesis promoter comprising at leastone of a chondroitin sulfate glucuronyltransferase, a gene encodingchondroitin sulfate glucuronyltransferase, a chondroitin sulfateN-acetylgalactosaminyltransferase-1, and a gene encoding a chondroitinsulfate N-acetylgalactosaminyltransferase-1.
 18. A treatment agent foran articular disease as described in claim 17, which promotes synthesisof chondroitin sulfate in cartilage.
 19. A cartilage repairing agentwhich can promote synthesis of chondroitin sulfate, the agentcomprising, as an active ingredient at least one of a chondroitinsulfate glucuronyltransferase, a gene encoding chondroitin sulfateglucuronyltransferase, a chondroitin sulfateN-acetylgalactosaminyltransferase-1, and a gene encoding a chondroitinsulfate N-acetylgalactosaminyltransferase-1.
 20. A cartilaginous tissueformation promoter comprising, as an active ingredient, at least one ofa chondroitin sulfate glucuronyltransferase, a gene encoding chondroitinsulfate glucuronyltransferase, a chondroitin sulfateN-acetylgalactosaminyltransferase-1, and a gene encoding a chondroitinsulfate N-acetylgalactosaminyltransferase-1.
 21. A method for promotingchondroitin sulfate synthesis in a living subject wherein the livingsubject includes a joint, a cartilage, an intervertebral disc or a cell,the method comprising the steps of: providing a gene comprising at leastone of a gene encoding chondroitin sulfate glucuronyltransferase and agene encoding chondroitin sulfate N-acetylgalactosaminyltransferase-1;and introducing the gene into the living subject.
 22. A method forpromoting a chondroitin sulfate synthesis in a living subject asdescribed in claim 21, wherein the step of introducing the gene into theliving subject is performed with a gene gun or via injection.
 23. Amethod for promoting a chondroitin sulfate synthesis in a living subjectwherein the living subject includes a joint, a cartilage, anintervertebral disc or a cell, the method comprising the steps of:providing at least one of chondroitin sulfate glucuronyltransferase andchondroitin sulfate N-acetylgalactosaminyltransferase-1; and introducingat least one of a chondroitin sulfate glucuronyltransferase andchondroitin sulfate N-acetylgalactosaminyltransferase-1 into the livingsubject.
 24. A method for promoting a chondroitin sulfate synthesis in aliving subject as described in claim 23, wherein the step of introducingat least one of a chondroitin sulfate glucuronyltransferase andchondroitin sulfate N-acetylgalactosaminyltransferase-1 into the livingsubject is performed via injection.
 25. A method for treating anarticular disease or discopathy, the method comprising the steps of:applying a therapeutically effective amount of an active ingredientcomprising at least one of a chondroitin sulfate glucuronyltransferase,a gene encoding chondroitin sulfate glucuronyltransferase, a chondroitinsulfate N-acetylgalactosaminyltransferase-1, and a gene encoding achondroitin sulfate N-acetylgalactosaminyltransferase-1 with theaffected joint or intervertebral disc; and promoting a chondroitinsulfate synthesis in the affected joint or intervertebral disc.